In an attempt to clarify the nature of rotavirus antigens and as a means of investigating rotavirus protein structure and function, we have isolated a series of monoclones directed at several rotavirus proteins. Monoclones with varying specificities were dereived from mice immunized with the Wa strain of human rotavirus (serotype 1), rhesus rotavirus (RRV) #2, or a reassortant rotavirus (ts bovine UK x DS-1) which has the human serotype 2 neutralization specificity. Monoclones were produced using standard hybridoma technology. Balb/C mice were immunized 3-4 times over the course of approximately 3 months with partially purified virus. The fusion ratio was 10 spleen cells per NS-1 myeloma cell. A solid phase radioimmunoassay (RIA) using either homologous or heterologous virus was the primary screening assay. Screening by hemagglutination-inhibition (HI) assay was a key adjunct to identifying monoclones directed at the major outer capsid protein of RRV. Positive samples were cloned once or twice by terminal dilution, grown in culture for study and inoculated into mice for production of acites fluid. Monoclonal antibodies were further characterized in immunoprecipitation experiments with viral lysates, in plaque reduction neutralization assays (PRNA), in solid phase RIA and HI assays using rassortant rotaviruses with predetermined genotype as antigens, and by immune electron microscopy (IEM).